World soybean production has grown 400% over the last 30 years. Consumer acceptance of soybean protein products has been growing and the perception of soybean products as a healthy food is strong. The number of new soy-protein-based products in the food marketplace has increased 11.2% per year for the past 3 years. The market has become more specific and the challenge is to produce new soy-protein-based food ingredients to enhance consumer acceptance and health.
Soybeans provide a good source of low-cost protein and have become an important world commodity because they are ubiquitous, have unique chemical composition, good nutritional value, versatile uses, and functional health benefits. Yet, less than about 5% of the soybean protein available is used for food, but this percentage is likely to grow. One of the main bodies of research in recent years has focused on studying individual storage proteins (glycinin and β-conglycinin) and relating them to industrially important functional properties and functional health benefits. In spite of this extensive research these individual proteins or enriched fractions thereof are not widely available at competitive costs.
Recently, there has been increased interest in obtaining purified β-conglycinin fractions, mainly to study its health beneficial properties such as cholesterol lowering and prevention of certain types of cancer. β-Conglycinin is a trimeric protein with a molecular weight of about 180 kDa. It is composed of three subunits: α′ (˜71 kDa), α(˜67 kDa), and β (˜50 kDa).
Geneticists have been trying, with mixed success, to develop soybean lines high in glycinin and others high in β-conglycinin. Soy protein isolates have >90% of protein on dry weight basis (N×6.25). Commercial yields of soy protein isolate are approximately 33% of the starting solids in defatted white flakes, corresponding to approximately 60% of the total protein recovered in the soy protein isolate (Sathe, 1989). In general, defatted soybean flour is extracted with water under alkaline conditions and then the extract is acidified to a pH between 4 and 5 to precipitate much of the protein. The precipitate collected is the isolated soy protein. This curd can be spray-dried or neutralized and spray-dried. Some soy protein isolate processes may use a combination of salts (Saio, 1975), reducing agents (Hirotsuka et al., 1988), electro-acidification (Bazinet et al., 2000), membrane filtration (Lawton et al., 1979), high-temperature short-time thermal treatments, but the details of these commercial processes are usually not fully disclosed and vary among manufacturers.
Soy proteins are not a homogeneous group. Soy proteins have been traditionally classified by their sedimentation coefficients as analyzed by ultracentrifugation into four large groups 2S (largely albumins and enzymes), 7S (largely β-conglycinin), 11S (largely glycinin), and 15S (largely dimers of glycinin) with peak molecular weights of approximately 25,000, 160,000, 350,000, and 600,000 respectively. A typical commercial process would yield approximately 22% 2S, 37% 7S, 31% 11S, and 11% 15S proteins as extracted by water, but these amounts may vary significantly depending on variety, crop year, handling and previous thermal treatment.
There have been complex laboratory procedures developed to fractionate these proteins from each other. Such techniques cannot be practically applied for commercial scale production. Some of these techniques are also difficult to reproduce because small variations in the procedures significantly alter the final composition of the fractions. There are also several patents filed for different fractionation processes, none of them are of practical use (Howard et al., 1981; Lehnhardt et al., 1983; Masahiko et al., 1994; Samoto et al., 1996; Savolainen, 1999; and Kohno et al., 2001).
Among the known methods developed for fractionating soybean proteins, one of the first attempts is a method using low temperatures (Wolf, 1956). Wolf recovered a fairly pure 11S fraction and used the name of cold-insoluble fraction (CIF) to define it. This method is further reported as cryoprecipitation and some authors name glycinin as the cryoprotein from soybeans (Wolf and Sly, 1967). These approaches are focused on the recovery of the 11S fraction and most of them do not discuss the 7S protein fraction.
Probably the most widely utilized procedure for fractionating glycinin and β-conglycinin is the one described by Than and Shibasaki in 1976. The fractionation of 7S and 11S globulins of soybean protein was achieved by extracting soybean meal with a Tris-buffer solution containing beta-mercaptoethanol at pH 7.8, centrifuging to remove the insoluble materials (primarily fiber), adjusting the pH of the supernatant to 6.6, dialysing, centrifuging to fractionate into crude 11S-rich precipitate and 7S-rich extract, adusting the pH to the isoelectric point (˜4.6) to precipitate the 7S-rich fraction, washing, and freeze-drying. To complete this purification of each fraction several column chromatography steps are required, which make this procedure far too expensive for industrial application.
The Japanese Patent Laid Open Publication No. 55010224 discloses a method utilizing the difference in reactivity with calcium wherein a small amount of calcium salt is added during extraction, allowing a 7S-rich fraction to be extracted. A similar method was reported by Saio et al. (1973, 1974, and 1975), where the calcium salt is added as the extraction buffer and the flour is first extracted to obtain a 7S-rich supernatant and the precipitate is redisolved and centrifuged to obtain a 11S-rich fraction. These methods have several drawbacks that make them impractical for industrial application. Beyond the processing drawbacks, the purities reported on an ultra-centrifugal basis (a less accurate method than we employ) are about 60%, which are lower than the purities obtained by our invention.
Further, other experimental methods to fractionate soy proteins have been reported by Roberts et al., 1965; Eldrige et al., 1967; Nagano et al., 1992; and Thiering et al., 2001. These methods for fractionating soy proteins are too expensive for industrial purposes, especially for the industrial production of soy protein isolates, and in some cases utilize chemicals that are not food-grade.
Recently, another method was developed by Saito et al. (2001), where the soy protein extract is treated with the enzyme phytase in order to hydrolyze phytic acid. They claim that by means of adjusting the pH, two fractions are obtained. The first fraction is rich in glycinin and the second rich in β-conglycinin. They claim that they obtain purities of about 80% for both fractions. The method requires incubation times and temperatures that make it impractical for industrial purposes and difficult to reproduce (Aldin, 2004). because of long process times and potential for microbiological growth and activity. Another drawback is that, it uses enzymes that add to the cost of the process.
Some attempts have been made to scale up some of these processes from the laboratory and to adapt them to pilot-plant production. Wu et al. (1999) successfully scaled up a method developed by Nagano et al. (1992) to obtain kg quantities of the individual soy storage proteins. But, the fraction yields are very low and the procedure is extremely costly and complicated for industrial production. Later, this procedure was improved (Rickert et al., 2003) obtaining three protein fractions, one β-conglycinin-rich, one glycinin-rich, and an intermediate mixture of the former two proteins plus a significant amount of lipoxygenase. Two waste stream products were produced, the spent flakes and the whey. This process was successfully scaled up to pilot-plant production, but its commercial application is questionable since it requires several extraction, centrifugation, and desalting steps.
It is therefore a primary objective of the present invention to provide a method of producing two separate vegetable protein isolates and/or products—one enriched for β-conglycinin and another enriched for glycinin.
It is another objective of the present invention to provide a method which avoids multistage fractionations steps and uses very small amounts of salts avoiding excessive washing and desalting steps.
It is still another objective to provide soy protein products that are highly functional in their performance as ingredients and, when possible, deliver high contents of naturally occurring healthy phytochemicals.
These and other objectives will become apparent from the following description.